Field of the Invention
The disclosure generally relates to mass spectrometry. More specifically, the disclosure relates to high resolution imaging mass spectrometry.
Description of the Related Art
Mass spectrometry (“MS”) is increasingly being used to analyze the composition of materials. Literature generally describes MS as an analytical technique that produces spectra of the masses of the atoms or molecules of a sample of material. The spectra are used to determine the elemental compositions or isotopic signatures of a sample, the masses of particles and of molecules, and if applicable, the chemical structures of molecules, such as peptides and other chemical compounds. MS works by ionizing compounds to generate charged molecules or molecule fragment ions and measuring their mass-to-charge (“m/z”) ratios, which can be correlated to a particular atom, molecule, or compound. In a typical MS procedure, a sample is first ionized. The sample may be solid, liquid, or gas. The ionization may for example, occur by bombarding the sample with electrons to cause some of the sample's molecules to break into charged molecules or fragments as ions. These ions are then separated according to their m/z ratio, typically by accelerating them and subjecting them to an electric or magnetic field, or combination of both fields. Motions of ions of the same m/z ratio as a species will be influenced similarly by the electric field (E), magnetic field (B), or E×B crossed fields, and undergo the same amount of deflection from their original trajectories. After ion separation, the ions are detected by a detector, which records the m/z for each species and results are displayed as spectra of the relative abundance of detected ions as a function of the m/z ratio. The atoms or molecules in the sample can be identified by correlating known masses to the identified masses or through a characteristic fragmentation pattern.
In MS, ionization refers to the production of gas phase ions suitable for resolution in the mass analyzer or mass filter. Although there are several ion sources that are available, ionization techniques have been described as belonging to either hard or soft ionization techniques. Hard ionization techniques include processes that impart high quantities of residual energy in the subject molecule and invoke large degrees of ion fragmentation. For singly-charged ions, resulting ions tend to have m/z ratios lower than the molecular mass. The most common example of hard ionization is Electron Impact (EI) ionization and other examples are Cf desorption and laser desorption.
Soft ionization refers to the processes which impart very little residual energy onto the subject molecule and thus result in very little ion fragmentation. Often, it is advantageous to avoid ion fragmentation to simplify the MS complexity and increase a signal-to-noise ratio for identification of an unknown substance through the identification of intact molecular ions. Examples include matrix-assisted laser desorption ionization (“MALDI”), electrospray ionization (“ESI”), chemical ionization (“CI”), and field desorption.
The literature also states that mass spectrometry imaging (also known as imaging mass spectrometry or “IMS”) is a technique used in mass spectrometry to visualize the spatial distribution of substances, such as compounds, biomarker, metabolites, peptides or proteins by their molecular masses. Emerging technologies in the field of IMS include MALDI imaging and secondary ion mass spectrometry (“SIMS”) imaging.
MALDI imaging techniques use a process in which a sample, typically a thin tissue section, is moved in front of a laser beam in two dimensions while the mass spectra are recorded. Generally. MALDI uses a matrix substance applied to a sample tissue. In MALDI, a laser ionizes the matrix substance applied to the sample tissue. A known issue with MALDI is that the application of the matrix substance to the sample tissue can cause migration of small cells and/or sample components that affect the accuracy of an analysis on a cell-by-cell resolution. Moreover, MALDI matrix may interact with surface chemicals and lead to bias analysis of certain classes of compounds. Further, a MALDI technique is generally only capable of probing in the 5-50 micro-meter (μm) range due to the large dimensions of the laser spot caused by a light diffraction limit.
However, single cell IMS for atoms or molecules within the cell components require substantially higher spatial resolving power. A cellular level analysis needs an order or two magnitude greater spatial resolution, such as in the nanometer range, than traditional MALDI provides. This high of a spatial resolution is beyond traditional MALDI's capabilities, although improvements are being made in MALDI's resolution to reduce laser ablation spot sizes to below 5 micron.
SIMS is used to analyze solid surfaces and thin films by sputtering the surface with a focused beam of ions (as primary ions) sometimes at high resolution of about 50 nano-meters, which causes ions of the sample to desorb. An analyzer collects and analyzes the ejected secondary ions of the sample. SIMS imaging is performed in a manner similar to electron microscopy; the primary ion beam is rastered across the sample while secondary mass spectra are recorded, SIMS is considered a hard ionization technique due to its significant energy imparted into the surface that causes fragmentation of ions. For some applications, the fragmentation is useful or at least acceptable, such as when analyzing metal atoms in a sample, where the molecules of the sample can be fragmented leaving the desired atom of the metal for further analysis. However, for sensitive or labile samples, such as proteins and other biological samples. SIMS can be too destructive to the sample to analyze various aspects of the sample.
Mile advances have been made in IMS, there are currently no known methods for desorbing intact molecules from smaller than one micron surfaces using soft ionization approaches. Although MALDI can desorb and concurrently ionize with a laser and an applied matrix for soft desorption, the resolution of a MALDI process is too large for cell-by-cell or particularly subcellular structures or other small samples that may be dislodged or otherwise desorbed for analysis by a mass spectrometer. SIMS has the capability of such small resolution, but with its hard ionization, can result in fragmentation that disrupts the structure of the molecule that is being sought to analyze.
Recent efforts have shown that radiofrequency can be used to ionize molecules. However, radio frequency ionization (“RFI”) may not have the resolution to directly desorb smaller selected portions of a sample.
There remains then a need to provide an improved system and method for mass spectrometry that allows soft desorption of a sample and subsequent ionization, but with sufficiently high image resolution to analyze sub-cellular sized structures.